Chemical Characterization of Wines Fermented with Various Malo-lactic Bacteria

Six malo-lactic strains of lactic acid bacteria were isolated from California wines and identified as Lactobacillus delbrueckii, L. buchneri, L. brevis, Leuconostoc citrovorum, and two strains of Pediococcus cerevisiae. Malo-lactic fermentation was induced in separate lots of wine by inoculation of each lot with one of the strains of bacteria. Malo-lactic fermentation had occurred in each inoculated wine within 2 months. The resultant wines were subjected to chemical analysis, including gas chromatographic examination of concentrated extracts of the wines. Only a few differences in composition were found when the malo-lactic wines were compared one with another. The differences that were found were in volatile acidity and in concentrations of acetoin (plus diacetyl) and probably diethyl succinate.     

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

Summary The amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources

Lacking an efficient process to produce 7-aminocephalosporanic acid from cephalosporin C in a single step, d-amino acid oxidase (DAAO) is of foremost importance in the industrial, two-step process used for this purpose. We report a detailed study on the catalytic properties of the three available DAAOs, namely, a mammalian DAAO and two others from yeast (Rhodotorula gracilis and Trigonopsis variabilis). In comparing the kinetic parameters determined for the three DAAOs, with both cephalosporin C and d-alanine as substrate, the catalytic efficiency of the two enzymes from microorganism is at least 2 orders of magnitude higher than that of pig kidney DAAO. Furthermore, the mammalian enzyme is more sensitive to product inhibition (from hydrogen peroxide and glutaryl-7-aminocephalosporanic acid). Therefore, enzymes from microorganisms appear to be by far more suitable catalysts for bioconversion, although some different minor differences are present between them (e.g., a higher activity of the R. gracilis enzyme when the bioconversion is carried out at saturating oxygen concentration). The mammalian DAAO, even being a poor catalyst, is more stable with respect to temperature than the R. gracilis enzyme in the free form. In any case, for industrial purposes DAAO is used only in the immobilized form where a strong enzyme stabilization occurs.     

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.     

Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Evolution of an acylase active on cephalosporin C

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.